Germplasm Enhancement with the First Medicinal Crop-Varieties for Prevention and Management of Cancer and Diabetes in Bitter Gourd (Momordica charantia)

Germplasm Enhancement with the First Medicinal Crop-Varieties for Prevention and Management of Cancer and Diabetes in Bitter Gourd (Momordica charantia) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Germplasm Enhancement with the First Medicinal Crop-Varieties for Prevention and Management of Cancer and Diabetes in Bitter Gourd (Momordica charantia) Chittaranjan Kole, Phullara Kole This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8161039/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Incidence of chronic diseases including cancer and diabetes is increasing globally. Fruits and vegetables are potential sources of functional phytochemicals for prevention and management of these diseases. Bitter gourd, Momordica charantia , a popular cucurbit crop, is known to contain a number of anticancer and antidiabetic phytomedicines but the popular cultivars in the available germplasm are poor in the content of these phytomedicines. We explored a wild bitter gourd germplasm and developed eight varieties including CBM3, CBM6, CBM10, CBM12, CBM13, CBM18, CBMH10 and CBMH12 with a view to broaden the genetic resources for nutraceuticals. We report here on the comparative contents of one anticancer phytomedicine, Cucurbitacin-B and two antidiabetic phytomedicines, Charantin and Plant Insulin in fresh fruits of 25 varieties in the enhanced germplasm constituting these eights medicinal varieties and 17 popular horticultural cultivars. We also report on their comparative performance with regard to fresh fruit weight, and correlation of the content of these three phytomedicines inter se and with fruit weight. Finally, we report on the identification of five varieties including CBMH10, CBMH12, CBM12, CBM10 and CBM13, in that order, for consumption of their fruits of as a source of functional foods for prevention and management of cancer and diabetes from the nutraceutically enhanced germplasm. Bitter gourd germplasm enhancement nutraceuticals medicinal crop-variety anticancer phytomedicine antidiabetic phytomedicine functional food Figures Figure 1 Figure 2 Figure 3 1. Introduction Prevalence of chronic diseases including cancer and diabetes is increasing globally (Giovannucci et al. 2010 ). There were about 20 million new cases of cancer with 9.7 million deaths due to it in 2022 (Bray et al. 2024 ). A recent study projects 18.5 million cancer deaths by 2050, an 89.7% increase over 2022 (Bizuayehu et al. 2025). Similarly, 589 million adults were living with diabetes in 2024 and the number of deaths due to it was 3.4 million ( https://diabetesatlas.org/ ). Death due to type-2 diabetes alone is projected at 3.68 million by 2050 (Chen et al. 2025 ). Utilization of nutraceutical crops, such as fruits and vegetables as a source of medicinally active phytochemicals is a potential alternative for prevention and management of these diseases (Mahmoud and Ibrahim 2021 ). Bitter gourd or bitter melon, Momordica charantia L, is one potential cucurbit vegetable crop that can be popularized as a functional food crop. This plant contains over 60 phytomedicines active against over 30 diseases (Raman and Lau 1996 ). Several medicinal properties of bitter gourd including antidiabetic, anticancer, antitumor, antileukemic, antioxidant, antiulcer, anti-inflammatory, hypocholesterolemic and hypotriglyceridemic properties are well documented in research (Behera et al. 2020 ). Almost all parts of bitter gourd, primarily the fruits, contain Cucurbitacin-B that have proven anticancer actions. They also contain Charantin and Plant Insulin (syn. p-Insulin, Polypeptide-p) that have clinically demonstrated hypoglycemic and antihyperglycemic activities and established beneficial effects on diabetes, especially of type-2. Plant Insulin can control both type I and type II diabetes (Chakraborty 2018 ). However, the popularly cultivated varieties of bitter gourd in the available germplasm have lower content of phytomedicines (Kole et al. 2010 ). We explored the wildly grown bitter gourd germplasm for development of varieties with high contents of these three anticancer and antidiabetic phytomedicines and developed eight varieties reported in this study. Since the weight of fresh fruits is also an important factor of total yield of a vegetable crop and the total content of phytomedicines in each fruit, we studied on the performance of these eight varieties along with 17 popularly grown varieties from seven countries with regard to both phytomedicine contents and fresh fruit weight, and also the nature and extent of correlation of the content of these three phytomedicines inter se and with fruit weight. This facilitated selection of the ideal varieties for consumption of their fruits as a source of functional foods for prevention and management of cancer and diabetes. 2. Materials and Methods 2.1. Plant Materials Six wildly grown plants belonging to Momordica charantia L. var. muricata , an allied botanical variety of the domesticated bitter gourd botanical variety, M.c. var. charantia were used for breeding of the first six medicinal varieties used in this study. Two of these medicinal varieties and a popularly grown variety, Taiwan White, belong to M.c. var. charantia were used for crossing to develop two dual-purpose hybrids. These eight varieties were grown under controlled conditions in a greenhouse following Complete Randomized Design with four replications along with 17 horticultural varieties grown popularly in seven Asian countries ( Supplementary Table 1 ). 2.2. Extraction and quantification of phytomedicines Quantification of the contents (mg/g) of one anticancer phytomedicine including Cucurbitacin-B and two antidiabetic phytomedicines including Charantin and Plant Insulin was done by HPLC analysis of lyophilized powders from fresh fruits of the 25 varieties in two replications. Detailed methods of extraction and quantification are described elsewhere (Kole et al. 2013 ). 2.3. Statistical analysis Statistical analysis of variation of phytomedicine contents was done by analysis of variance (ANOVA) following Completely Randomized Design (CRD). The varietal effects were compared post hoc using Duncan’s Multiple Range Test (DMRT) and assigned alphabetical grouping ranks. Correlation analysis was done following routine statistical analysis. 3. Results 3.1. Enhancement of germplasm with medicinal varieties Pure-line selection from six wildly grown plants belonging to Momordica charantia var. muricata , an allied botanical variety of M.c. var. charantia to which most of the popularly grown horticultural varieties of bitter gourd belong led to the development of six bitter gourd varieties including CBM3, CBM6, CBM10, CBM12, CBM13 and CBM18. Preliminary studies exhibited smaller fruit size and higher phytomedicine contents of these varieties as compared to the popular cultivars. Two varieties, CBM10 and CBM12, selected from them based on higher phytomedicine contents were hybridized with Taiwan White, a popularly grown cultivated variety (cultivar) with the largest fruit size belonging to M.c. charantia var. charantia that resulted in two hybrid varieties, CBMH10 and CBMH12. Results of evaluation of these eight varieties with regard to phytomedicine contents and fresh fruit weight are presented below. 3.2. Variation in phytomedicine contents in the enhanced germplasm Statistical analysis of the contents of the three phytomedicines as mg/g of lyophilized powers of fresh fruits of the 25 bitter gourd varieties including the eight CBM varieties and 17 popular cultivars revealed highly significant variation (Fig. 1 , Supplementary Table 1). The contents of Cucurbitacin-B were significantly the highest in CBM10 (0.645mg/g) and CBMH10 (0.615mg) followed by CBM13 (0.5mg). Two popular cultivars, Hybrid White Pearl and Taiwan White were equally the lowest performing varieties containing 0.12mg and 0.13mg, respectively. With respect to Charantin, CBMH10 (8.0mg) again exhibited significantly the highest content closely followed by CBM12 (7.88mg). Hybrid India Star (4.2mg) followed by Hybrid Baby Doll (5.37mg) had the lowest Charantin content. CBMH10 (4.0mg) had the highest content of Plant Insulin and was seconded by CBMH12 (0.3mg) that was closely followed by CBM12 and CBM3 with similar (0.26mg) content. Six cultivars including Hybrid Beauty Winner, Hong Kong Green, Taiwan White, Hybrid Bangkok Large, Hybrid India Star and Hybrid Taiwan White had equally the least (0.11-0.12mg) contents. 3.3. Variation in fruit sizes in the enhanced germplasm Fresh fruit weight also exhibited highly significant variation among the varieties (Fig. 2 , Supplementary Table 1 ). The varieties belonging to M.c. charantia bore, in general, larger fruits weighing from 60.6g to 146.33g. The M.c. muricata varieties had varied sizes ranging from 3.48g to 78.59g. The six CBM varieties bore smaller fruit weighing from 3.48g to 41.67g. Statistically six varieties including CBM12 (3.84g), CBM10 (4.90g) and CBM18 (5.89g) had the smallest fruits whereas Taiwan White (146.33g) had by far the largest fruits among all. Interestingly, the two hybrid varieties, CBMH12 (74.36g) and CBMH10 (64.47g) had moderate sized fruits ( Supplementary Fig. 1 ). 3.4. Correlations among phytomedicine contents fruit weight Correlation analysis among the contents of three phytomedicines and fruit weight revealed highly significant positive association ( r = 0.672) between cucurbitacin-B content and Plant Insulin content. Charantin content and Plant Insulin content were also highly and positively correlated ( r = 0.518). Interestingly, fruit weight had highly significant negative association with Cucurbitacin-B ( r = -0.65). Correlations of other combinations were statistically non-significant (Fig. 3 , Supplementary Table 2 ). 4. Discussion The eight bitter gourd varieties of the CBM series had, in general, higher contents of all the three anticancer and antidiabetic phytomedicines in comparison to the 17 popularly grown cultivars belonging to both M.c muricata and M.c. charantia botanical varieties. Among them, CBMH10 was found to be universally the richest. CBM12 ranked second for Charantin content and third for Plant Insulin content. Other three varieties including CBMH12, CBM3 and CBM13 had also considerable content of the phytomedicines. Therefore, consumption of fresh fruits of the varieties of the CBM series, particularly the above five varieties and their extracts or powders may help in mitigation of the problems of cancer and diabetes. Since diabetes leads also to blindness, kidney failure, heart attacks, stroke and lower limb amputation ( https://www.who.int/news-room/fact-sheets/detail/noncommunicable-diseases ), use of these varieties as functional foods may help in controlling these diseases. Fruit weight is an important factor of fruit yield and total phytomedicine contained in fruits. The CBM varieties had, in general, smaller fruits as compared to the popular cultivars. However, the two hybrids CBMH12 and CBMH10 bore heavier fruits equaling to many popular cultivars. These two hybrids, therefore, emerged as superior to all the varieties considering both medicinal and horticultural traits and may be recommended as dual-purpose varieties. The correlation study has led to an interesting observation of negative correlation between fruit weight and Cucurbitacin-B content indicating the requirement of compromising fruit yield for utilization of bitter gourd varieties as a source of this anticancer phytomedicine. Another interesting observation was the positive correlation of the contents of the two antidiabetic phytomedicines, Charantin and Plant Insulin suggesting future studies on genetic and biochemical mechanisms underlying their production. All the CBM varieties were developed by either selection or hybridization, both traditional breeding methods, and so their fruits and fruit products can be safely consumed and these varieties may enjoy acceptance by both the farmers and consumers. 5. Conclusion The results from this study demonstrates the importance of developing crop varieties with higher contents of medicinally active bioactives for prevention and management of chronic and fatal diseases for their use as sources of functional foods. They also evidenced that wild germplasm could be a potential genetic resource for developing functional food varieties in crops. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Funding The first author acknowledges USDA-SAC for financial support for the work. The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Author Contribution Both the authors contributed to the study conception and design; material preparation, data collection and analysis; and manuscript preparation. Both the authors read and approved the final manuscript. Acknowledgement The authors acknowledge Prof. (Rtd.) Albert G. Abbot of the Department of Genetics and Biochemistry and Prof. (Rtd.) Richard K Marcus of the Department of Chemistry, Clemson University, Clemson, SC, USA for laboratory facilities; Dr. Bode Olukolu for assistance in extraction and quantification of phytomedicines –while working at the Department of Genetics and Biochemistry, and Prof. Deb Sankar Gupta from the Uttar Banga Krishi Viswavidyalaya, India for assistance in statistical analysis. Data Availability The detailed data are not publicly available but we will be happy to share data on reasonable request. References Giovannucci E, Harlan DM, Archer MC, Bergenstal RM, Gapstur SM, Habel LA et al. (2010) Diabetes and cancer - a consensus report. Diab Care 33(7): 1674–1685. doi: 10.2337/dc10-0666 . Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, Jemal A (2024) Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 74(3): 229–263. doi: 10.3322/caac.21834. Epub 2024 Apr 4. PMID: 38572751. Bizuayehu HM, Ahmed KY, Kibret GD, Dadi AF, Belachew SA, Bagade T, Tegegne T K, Venchiarutti RL, Kibret KT, Hailegebireal AH, Assefa Y, Khan MN, Abajobir A, Alene KA, Mengesha Z, Erku D, Enquobahrie DA, Minas TZ, Misgan E, Ross AG (2024) Global disparities of cancer and its projected burden in 2050. JAMA Netw Open 7(11):e2443198. doi: 10.1001/jamanetworkopen.2024.43198 Chen Y, Wang G, Hou Z, Liu X, Ma S, Jiang M (2025) Comparative diabetes mellitus burden trends across global, Chinese, US, and Indian populations using GBD 2021 database. Sci Rep 15: 11955. doi.org/10.1038/s41598-025-96175-4 . Mahmoud S, Ibrahim AAA (2021) Fruits and vegetables as sources of functional phytochemicals for the prevention and management of obesity, diabetes, and cancer. In: Egbuna C, Hassan S (eds) Dietary Phytochemicals, Springer Nature Switzerland AG, Cham, Switzerland, pp 147–167). doi: 10.1007/978-3-030-72999-8_8 . Raman A, Lau C (1996) Anti-diabetic properties and phytochemistry of Momordica charantia L. (Cucurbitaceae). Phytomedicine 2(4): 349 – 262. doi: 10.1016/S0944-7113(96)80080-8 . Behera TK, Matsumura H, Kole C (2020) Glimpse on genomics and breeding in bitter gourd: a crop of the future for food, nutrition and health security. In: Kole C, Matsumura H, Behera TK (eds) The bitter gourd genome, Springer Nature Switzerland AG, Cham, Switzerland, pp 1–6. doi.org/10.1007/978-3-030-15062-4 . Chakraborty P (2018) Development of new anti-diabetic drug from medicinal plant-genomic research. J Diabetol 2(1): 6–7. Kole C, Olukolu BA, Kole P, Abbott AG (2010) Association mapping of fruit traits and phytomedicine contents in a structured population of bitter melon ( Momordica charantia L.). In: Thies JA, Kousik S, Levi A (eds) Cucurbitaceae 2010 Proceedings. Amer Soc Hortic Sci, Alexandria, VA, pp 42–45. Kole C, Kole P, Randunu KM, Choudhary P, Podila R, Ke PC, Rao AM, Marcus RK (2013) Nanobiotechnology can boost crop production and quality: first evidence from increased plant biomass, fruit yield and phytomedicine content in bitter melon ( Momordica charantia ). BMC Biotechnol 13: 37. Doi: 10.1186/1472-6750-13-37 . Additional Declarations No competing interests reported. Supplementary Files KoleKoleSupplementaryInformation.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8161039","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":550985538,"identity":"2bbe1915-00a3-4a89-bc27-8af534a96d23","order_by":0,"name":"Chittaranjan 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09:54:34","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":46105,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8161039/v1/98adccdd1f2bcad05ec26b12.html"},{"id":96984595,"identity":"ff4110dc-fe01-4c18-9125-1a3dcd7a1d42","added_by":"auto","created_at":"2025-11-28 09:54:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":129391,"visible":true,"origin":"","legend":"\u003cp\u003eComparative contents of Cucurbitacin-B (1a) and Charantin (1b) and Plant Insulin (1c) in 25 bitter gourd varieties of the enhanced germplasm. The vertical lines (error bars) indicate mean±std on top of each bar in the figure. Details about the genotypes are mentioned in Supplementary Table 1.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8161039/v1/43e43b872475fd56669c00ec.png"},{"id":96984602,"identity":"96943c92-f7cd-4714-ad76-c74028a89c8e","added_by":"auto","created_at":"2025-11-28 09:54:34","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":94287,"visible":true,"origin":"","legend":"\u003cp\u003eVariation in fruit weights among 25 varieties of bitter gourd in the enhanced germplasm. The vertical lines (error bars) indicate mean±std on top of each bar in the figure. Details about the genotypes are mentioned in Supplementary Table 1.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8161039/v1/151ef2ab3b7918ec37f07403.jpg"},{"id":96984613,"identity":"89af5823-7475-4330-9acc-ac4571eada26","added_by":"auto","created_at":"2025-11-28 09:54:34","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":35972,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation of the contents of Cucurbitacin-B (B), Charantin (CHR) and Plant Insulin (PLIN) \u003cem\u003einter se\u003c/em\u003e and with fruit weight (Fr Wt) in bitter gourd. Correlation coefficient (\u003cem\u003er\u003c/em\u003e) values at the center.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8161039/v1/b5c46ef61c38efdc1d8616af.jpg"},{"id":99277192,"identity":"6ea0aeb0-269d-439f-9c28-e3a067ef25fa","added_by":"auto","created_at":"2025-12-31 07:24:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":776844,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8161039/v1/dbfe66f3-1cea-426d-a741-4bc08ce48a50.pdf"},{"id":96984603,"identity":"8aba4cd0-cb25-4c3c-9907-cd672da67564","added_by":"auto","created_at":"2025-11-28 09:54:34","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":225669,"visible":true,"origin":"","legend":"","description":"","filename":"KoleKoleSupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8161039/v1/e0f76b3c35d264127cfbd8f1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Germplasm Enhancement with the First Medicinal Crop-Varieties for Prevention and Management of Cancer and Diabetes in Bitter Gourd (Momordica charantia)","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePrevalence of chronic diseases including cancer and diabetes is increasing globally (Giovannucci et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). There were about 20\u0026nbsp;million new cases of cancer with 9.7\u0026nbsp;million deaths due to it in 2022 (Bray et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). A recent study projects 18.5\u0026nbsp;million cancer deaths by 2050, an 89.7% increase over 2022 (Bizuayehu et al. 2025). Similarly, 589\u0026nbsp;million adults were living with diabetes in 2024 and the number of deaths due to it was 3.4\u0026nbsp;million (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://diabetesatlas.org/\u003c/span\u003e\u003cspan address=\"https://diabetesatlas.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Death due to type-2 diabetes alone is projected at 3.68\u0026nbsp;million by 2050 (Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Utilization of nutraceutical crops, such as fruits and vegetables as a source of medicinally active phytochemicals is a potential alternative for prevention and management of these diseases (Mahmoud and Ibrahim \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBitter gourd or bitter melon, \u003cem\u003eMomordica charantia\u003c/em\u003e L, is one potential cucurbit vegetable crop that can be popularized as a functional food crop. This plant contains over 60 phytomedicines active against over 30 diseases (Raman and Lau \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Several medicinal properties of bitter gourd including antidiabetic, anticancer, antitumor, antileukemic, antioxidant, antiulcer, anti-inflammatory, hypocholesterolemic and hypotriglyceridemic properties are well documented in research (Behera et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Almost all parts of bitter gourd, primarily the fruits, contain Cucurbitacin-B that have proven anticancer actions. They also contain Charantin and Plant Insulin (syn. p-Insulin, Polypeptide-p) that have clinically demonstrated hypoglycemic and antihyperglycemic activities and established beneficial effects on diabetes, especially of type-2. Plant Insulin can control both type I and type II diabetes (Chakraborty \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, the popularly cultivated varieties of bitter gourd in the available germplasm have lower content of phytomedicines (Kole et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). We explored the wildly grown bitter gourd germplasm for development of varieties with high contents of these three anticancer and antidiabetic phytomedicines and developed eight varieties reported in this study.\u003c/p\u003e\u003cp\u003eSince the weight of fresh fruits is also an important factor of total yield of a vegetable crop and the total content of phytomedicines in each fruit, we studied on the performance of these eight varieties along with 17 popularly grown varieties from seven countries with regard to both phytomedicine contents and fresh fruit weight, and also the nature and extent of correlation of the content of these three phytomedicines \u003cem\u003einter se\u003c/em\u003e and with fruit weight. This facilitated selection of the ideal varieties for consumption of their fruits as a source of functional foods for prevention and management of cancer and diabetes.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Plant Materials\u003c/h2\u003e\u003cp\u003eSix wildly grown plants belonging to \u003cem\u003eMomordica charantia\u003c/em\u003e L. var. \u003cem\u003emuricata\u003c/em\u003e, an allied botanical variety of the domesticated bitter gourd botanical variety, \u003cem\u003eM.c.\u003c/em\u003e var. \u003cem\u003echarantia\u003c/em\u003e were used for breeding of the first six medicinal varieties used in this study. Two of these medicinal varieties and a popularly grown variety, Taiwan White, belong to \u003cem\u003eM.c.\u003c/em\u003e var. \u003cem\u003echarantia\u003c/em\u003e were used for crossing to develop two dual-purpose hybrids. These eight varieties were grown under controlled conditions in a greenhouse following Complete Randomized Design with four replications along with 17 horticultural varieties grown popularly in seven Asian countries (\u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Extraction and quantification of phytomedicines\u003c/h2\u003e\u003cp\u003eQuantification of the contents (mg/g) of one anticancer phytomedicine including Cucurbitacin-B and two antidiabetic phytomedicines including Charantin and Plant Insulin was done by HPLC analysis of lyophilized powders from fresh fruits of the 25 varieties in two replications. Detailed methods of extraction and quantification are described elsewhere (Kole et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Statistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis of variation of phytomedicine contents was done by analysis of variance (ANOVA) following Completely Randomized Design (CRD). The varietal effects were compared post hoc using Duncan\u0026rsquo;s Multiple Range Test (DMRT) and assigned alphabetical grouping ranks. Correlation analysis was done following routine statistical analysis.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Enhancement of germplasm with medicinal varieties\u003c/h2\u003e\u003cp\u003ePure-line selection from six wildly grown plants belonging to \u003cem\u003eMomordica charantia\u003c/em\u003e var. \u003cem\u003emuricata\u003c/em\u003e, an allied botanical variety of \u003cem\u003eM.c.\u003c/em\u003e var. \u003cem\u003echarantia\u003c/em\u003e to which most of the popularly grown horticultural varieties of bitter gourd belong led to the development of six bitter gourd varieties including CBM3, CBM6, CBM10, CBM12, CBM13 and CBM18. Preliminary studies exhibited smaller fruit size and higher phytomedicine contents of these varieties as compared to the popular cultivars. Two varieties, CBM10 and CBM12, selected from them based on higher phytomedicine contents were hybridized with Taiwan White, a popularly grown cultivated variety (cultivar) with the largest fruit size belonging to \u003cem\u003eM.c. charantia\u003c/em\u003e var. \u003cem\u003echarantia\u003c/em\u003e that resulted in two hybrid varieties, CBMH10 and CBMH12. Results of evaluation of these eight varieties with regard to phytomedicine contents and fresh fruit weight are presented below.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Variation in phytomedicine contents in the enhanced germplasm\u003c/h2\u003e\u003cp\u003eStatistical analysis of the contents of the three phytomedicines as mg/g of lyophilized powers of fresh fruits of the 25 bitter gourd varieties including the eight CBM varieties and 17 popular cultivars revealed highly significant variation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cb\u003eSupplementary Table\u0026nbsp;1).\u003c/b\u003e The contents of Cucurbitacin-B were significantly the highest in CBM10 (0.645mg/g) and CBMH10 (0.615mg) followed by CBM13 (0.5mg). Two popular cultivars, Hybrid White Pearl and Taiwan White were equally the lowest performing varieties containing 0.12mg and 0.13mg, respectively. With respect to Charantin, CBMH10 (8.0mg) again exhibited significantly the highest content closely followed by CBM12 (7.88mg). Hybrid India Star (4.2mg) followed by Hybrid Baby Doll (5.37mg) had the lowest Charantin content. CBMH10 (4.0mg) had the highest content of Plant Insulin and was seconded by CBMH12 (0.3mg) that was closely followed by CBM12 and CBM3 with similar (0.26mg) content. Six cultivars including Hybrid Beauty Winner, Hong Kong Green, Taiwan White, Hybrid Bangkok Large, Hybrid India Star and Hybrid Taiwan White had equally the least (0.11-0.12mg) contents.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.3. Variation in fruit sizes in the enhanced germplasm\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eFresh fruit weight also exhibited highly significant variation among the varieties (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e). The varieties belonging to \u003cem\u003eM.c. charantia\u003c/em\u003e bore, in general, larger fruits weighing from 60.6g to 146.33g. The \u003cem\u003eM.c. muricata\u003c/em\u003e varieties had varied sizes ranging from 3.48g to 78.59g. The six CBM varieties bore smaller fruit weighing from 3.48g to 41.67g. Statistically six varieties including CBM12 (3.84g), CBM10 (4.90g) and CBM18 (5.89g) had the smallest fruits whereas Taiwan White (146.33g) had by far the largest fruits among all. Interestingly, the two hybrid varieties, CBMH12 (74.36g) and CBMH10 (64.47g) had moderate sized fruits (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Correlations among phytomedicine contents fruit weight\u003c/h2\u003e\u003cp\u003eCorrelation analysis among the contents of three phytomedicines and fruit weight revealed highly significant positive association (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.672) between cucurbitacin-B content and Plant Insulin content. Charantin content and Plant Insulin content were also highly and positively correlated (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.518). Interestingly, fruit weight had highly significant negative association with Cucurbitacin-B (\u003cem\u003er\u003c/em\u003e = -0.65). Correlations of other combinations were statistically non-significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cb\u003eSupplementary Table\u0026nbsp;2\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe eight bitter gourd varieties of the CBM series had, in general, higher contents of all the three anticancer and antidiabetic phytomedicines in comparison to the 17 popularly grown cultivars belonging to both \u003cem\u003eM.c muricata\u003c/em\u003e and \u003cem\u003eM.c. charantia\u003c/em\u003e botanical varieties. Among them, CBMH10 was found to be universally the richest. CBM12 ranked second for Charantin content and third for Plant Insulin content. Other three varieties including CBMH12, CBM3 and CBM13 had also considerable content of the phytomedicines. Therefore, consumption of fresh fruits of the varieties of the CBM series, particularly the above five varieties and their extracts or powders may help in mitigation of the problems of cancer and diabetes. Since diabetes leads also to blindness, kidney failure, heart attacks, stroke and lower limb amputation (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.who.int/news-room/fact-sheets/detail/noncommunicable-diseases\u003c/span\u003e\u003cspan address=\"https://www.who.int/news-room/fact-sheets/detail/noncommunicable-diseases\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), use of these varieties as functional foods may help in controlling these diseases.\u003c/p\u003e\u003cp\u003eFruit weight is an important factor of fruit yield and total phytomedicine contained in fruits. The CBM varieties had, in general, smaller fruits as compared to the popular cultivars. However, the two hybrids CBMH12 and CBMH10 bore heavier fruits equaling to many popular cultivars. These two hybrids, therefore, emerged as superior to all the varieties considering both medicinal and horticultural traits and may be recommended as dual-purpose varieties.\u003c/p\u003e\u003cp\u003eThe correlation study has led to an interesting observation of negative correlation between fruit weight and Cucurbitacin-B content indicating the requirement of compromising fruit yield for utilization of bitter gourd varieties as a source of this anticancer phytomedicine. Another interesting observation was the positive correlation of the contents of the two antidiabetic phytomedicines, Charantin and Plant Insulin suggesting future studies on genetic and biochemical mechanisms underlying their production.\u003c/p\u003e\u003cp\u003eAll the CBM varieties were developed by either selection or hybridization, both traditional breeding methods, and so their fruits and fruit products can be safely consumed and these varieties may enjoy acceptance by both the farmers and consumers.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe results from this study demonstrates the importance of developing crop varieties with higher contents of medicinally active bioactives for prevention and management of chronic and fatal diseases for their use as sources of functional foods. They also evidenced that wild germplasm could be a potential genetic resource for developing functional food varieties in crops.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe first author acknowledges USDA-SAC for financial support for the work. The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eBoth the authors contributed to the study conception and design; material preparation, data collection and analysis; and manuscript preparation. Both the authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors acknowledge Prof. (Rtd.) Albert G. Abbot of the Department of Genetics and Biochemistry and Prof. (Rtd.) Richard K Marcus of the Department of Chemistry, Clemson University, Clemson, SC, USA for laboratory facilities; Dr. Bode Olukolu for assistance in extraction and quantification of phytomedicines \u0026ndash;while working at the Department of Genetics and Biochemistry, and Prof. Deb Sankar Gupta from the Uttar Banga Krishi Viswavidyalaya, India for assistance in statistical analysis.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe detailed data are not publicly available but we will be happy to share data on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGiovannucci E, Harlan DM, Archer MC, Bergenstal RM, Gapstur SM, Habel LA et al. (2010) Diabetes and cancer - a consensus report. 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In: Kole C, Matsumura H, Behera TK (eds) The bitter gourd genome, Springer Nature Switzerland AG, Cham, Switzerland, pp 1\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003edoi.org/10.1007/978-3-030-15062-4\u003c/span\u003e\u003cspan address=\"10.1007/978-3-030-15062-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChakraborty P (2018) Development of new anti-diabetic drug from medicinal plant-genomic research. J Diabetol 2(1): 6\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKole C, Olukolu BA, Kole P, Abbott AG (2010) Association mapping of fruit traits and phytomedicine contents in a structured population of bitter melon (\u003cem\u003eMomordica charantia\u003c/em\u003e L.). In: Thies JA, Kousik S, Levi A (eds) Cucurbitaceae 2010 Proceedings. Amer Soc Hortic Sci, Alexandria, VA, pp 42\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKole C, Kole P, Randunu KM, Choudhary P, Podila R, Ke PC, Rao AM, Marcus RK (2013) Nanobiotechnology can boost crop production and quality: first evidence from increased plant biomass, fruit yield and phytomedicine content in bitter melon (\u003cem\u003eMomordica charantia\u003c/em\u003e). BMC Biotechnol 13: 37. Doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/1472-6750-13-37\u003c/span\u003e\u003cspan address=\"10.1186/1472-6750-13-37\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bitter gourd, germplasm enhancement, nutraceuticals, medicinal crop-variety, anticancer phytomedicine, antidiabetic phytomedicine, functional food","lastPublishedDoi":"10.21203/rs.3.rs-8161039/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8161039/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIncidence of chronic diseases including cancer and diabetes is increasing globally. Fruits and vegetables are potential sources of functional phytochemicals for prevention and management of these diseases. Bitter gourd, \u003cem\u003eMomordica charantia\u003c/em\u003e, a popular cucurbit crop, is known to contain a number of anticancer and antidiabetic phytomedicines but the popular cultivars in the available germplasm are poor in the content of these phytomedicines. We explored a wild bitter gourd germplasm and developed eight varieties including CBM3, CBM6, CBM10, CBM12, CBM13, CBM18, CBMH10 and CBMH12 with a view to broaden the genetic resources for nutraceuticals. We report here on the comparative contents of one anticancer phytomedicine, Cucurbitacin-B and two antidiabetic phytomedicines, Charantin and Plant Insulin in fresh fruits of 25 varieties in the enhanced germplasm constituting these eights medicinal varieties and 17 popular horticultural cultivars. We also report on their comparative performance with regard to fresh fruit weight, and correlation of the content of these three phytomedicines \u003cem\u003einter se\u003c/em\u003e and with fruit weight. Finally, we report on the identification of five varieties including CBMH10, CBMH12, CBM12, CBM10 and CBM13, in that order, for consumption of their fruits of as a source of functional foods for prevention and management of cancer and diabetes from the nutraceutically enhanced germplasm.\u003c/p\u003e","manuscriptTitle":"Germplasm Enhancement with the First Medicinal Crop-Varieties for Prevention and Management of Cancer and Diabetes in Bitter Gourd (Momordica charantia)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-28 09:54:29","doi":"10.21203/rs.3.rs-8161039/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0d19e895-08d2-4162-b185-098af8ecb844","owner":[],"postedDate":"November 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-31T07:23:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-28 09:54:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8161039","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8161039","identity":"rs-8161039","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}